Anti-Leukemic Effects of Velcrin in SLFN12-Expressing Acute Myeloid Leukemia

Schlafen 12 (SLFN12) is a member of the Schlafen (SLFN) family of proteins, a group of interferon-stimulated genes with diverse roles in cellular regulation with implications for human malignancies. Accumulating evidence indicates that SLFN family proteins may serve as prognostic markers across various cancer types. In acute myeloid leukemia (AML), SLFN12 is notably overexpressed, which prompted us to investigate its potential as a therapeutic target. Employing a panel of leukemia cell lines, we explored the effects of velcrins, a class of small molecules able to modulate SLFN12 biological activity. Mechanistic studies showed that velcrin treatment increases expression of SLFN12 and promotes SLFN12 complex formation with PDE3A or PDE3B. Functionally, these effects were associated with growth inhibition and induction of apoptosis. Further, velcrin treatment induced potent suppressive effects on the clonogenic capability of primary human AML progenitors and suppressed tumor growth and significantly extended survival in a mouse AML xenograft model. Taken together, these findings highlight the potential of using velcrins as a promising therapeutic strategy for the treatment of AML patients. Animal experiment protocols were approved by the Northwestern University Institutional Animal Care and Use Committee and performed accordingly. 5–6-week-old NU/NU nude female mice were acquired from Charles River laboratories (Strain Code 088). To induce flank tumors, 5x106 early passage HEL cells in the logarithmic growth phase were collected, washed, and resuspended in a final volume of 100 µL in PBS and loaded into a U-100 insulin syringe with an attached 27-gauge needle and injected into the right flank. Approximately 13 days after injection, once tumors were palpable, tumor measurements were collected and mice were randomized into two treatment groups: vehicle control or treatment with a velcrin (BAY 2666605), with 5 mice per group. Mice were treated with vehicle control or BAY 2666605 (5 mg/kg), which was administered orally, twice daily for 2 weeks, with two days off. Tumor size was measured three times per week by caliper and the formula was calculated as follows (D × d2)* π/6, where D is the longest diameter and d is the shorter diameter. Body weights and clinical observations were recorded 3 times per week. For survival, mice were observed daily and all animals were compassionately euthanized 30 minutes following the last treatment dose. Tumor sections (n=4 per experimental group) were subsequently processed for SDS page or RNA-seq analysis.

Schlafen 12（SLFN12）属于Schlafen（SLFN）蛋白家族，该家族为一类干扰素刺激基因（interferon-stimulated genes），在细胞调控中发挥多种功能，且与人类恶性肿瘤的发生发展密切相关。越来越多的研究证据表明，SLFN家族蛋白可作为多种癌症类型的预后标志物。在急性髓系白血病（acute myeloid leukemia, AML）中，SLFN12呈现显著高表达，这促使我们探究其作为治疗靶点的潜力。本研究利用一组白血病细胞系，探究了velcrins（一类可调控SLFN12生物学活性的小分子化合物）的生物学效应。机制研究显示，velcrin处理可上调SLFN12的表达，并促进SLFN12与磷酸二酯酶3A（PDE3A）、磷酸二酯酶3B（PDE3B）形成复合物。功能层面上，这些效应与细胞生长抑制及凋亡诱导相关。此外，velcrin处理可对原代人AML祖细胞的克隆形成能力产生强效抑制作用，同时在小鼠AML异种移植模型中可抑制肿瘤生长并显著延长模型小鼠的生存期。综上，本研究结果表明，velcrins有望成为治疗AML患者的潜在治疗策略。

本研究的动物实验方案已获得西北大学机构动物护理与使用委员会（Northwestern University Institutional Animal Care and Use Committee）批准，并严格按照方案执行。实验所用的5~6周龄雌性NU/NU裸鼠购自Charles River实验室（品系代码：088）。为构建皮下移植瘤模型，收集处于对数生长期的早期传代HEL细胞，经洗涤后重悬于100 μL磷酸盐缓冲液（phosphate buffered saline, PBS）中，使用装有27G针头的U-100胰岛素注射器将细胞悬液注射至小鼠右侧背部皮下。细胞接种约13天后，待肿瘤可触及后，测量肿瘤体积并将小鼠随机分为两组：溶剂对照组与velcrin（BAY 2666605）处理组，每组5只小鼠。两组小鼠分别接受溶剂对照或BAY 2666605（5 mg/kg）灌胃处理，每日给药2次，连续给药2周后停药2天。每周使用游标卡尺测量三次肿瘤大小，肿瘤体积计算公式为：(D × d²) × π/6，其中D为肿瘤最长径，d为肿瘤最短径。每周记录三次小鼠体重与临床状态。生存分析组小鼠每日接受观察，末次给药30分钟后对所有小鼠实施安乐死。随后，取每组4个肿瘤组织样本，分别进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE）与RNA测序（RNA sequencing, RNA-seq）分析。